Development of a new animal cell bioreactor for monoclonal antibody production | |
| Author | Suthasinee Nilsaeng |
| Call Number | AIT Diss. no.FB-07-02 |
| Subject(s) | Monocronal antibodies Animal cell biotechnology |
| Note | A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Food Engineering and Bioprocess Technology, School of Environment, Resources and Development |
| Publisher | Asian Institute of Technology |
| Series Statement | Dissertation ; no. FB-07-02 |
| Abstract | Today the biotechnology and pharmaceutical industrial sectors are increasingly focusing on mammalian cell culture for bio-production of various therapeutics including antibodies, hormones, interferons, erythropoietin, clotting factor, immunoadhesins and vaccines. The market for monoclonal antibodies (mAbs) is expected to grow 30% a year. This demand has motivated and provided a challenge for developing efficient and cost effective processes for mAb production. High cell density and good long-term culture stability are two key factors in developing a continuous mAb production. This dissertation focuses on development a novel bioreactor for long term mAb production and enhancement cell growth and its productivity. In developing a bioreactor for long term mAb production, a novel supermacroporous cryogel matrix was selected for use as a supporting material for immobilized hybridoma cells. In the first section of this research, a supermacroporous cryogel monolith reactor has been developed to culture hybridoma cells for long term continuous production of mAb. Hybridoma clone M2139 which produces IgG2b antibodies specific for Jl epitope (GERGAAGIAGPK; amino acids, 551-564) of collagen type II and induced arthritis in mice was studied in this work. This model system is used for further diagnoses and study of arthritis. The hybridoma cells were immobilized in the porous matrix of a cryogel monolith column (10 ml bed volume) over a period of 48 h. Cells were in the lag phase for 15 days and secreted mAb into the circulation medium. The kinetics of glucose consumption, lactic acid and mAb production were studied. The mAb concentration reached 130 ug/ml after continuous recirculation in the cryogel monolith column for 36 days. The yield of the mAb after purification was 67.5 mg/l, which was three times greater than mAb yield obtained from T-flask batch cultivation. Even after a fresh medium reservoir was replaced, cells in cryogel monolith column were still active and had relatively stable mAb production for an extended period of time. The bioreactor was operated continuously for a total of 55 days. The results from ELISA as well as arthritis experiments demonstrate that the antibodies secreted by cells grown on the cryogel monolith column did not differ from antibodies purified from the cells grown in commercial CL- 1000 culture flasks. For enhancing cell growth and its productivity, the effects of basal medium and aketoglutarate on different hybridoma clone were studied in the second section. The experiment was carried out in two-dimensional (2-D) cultures on multi-well plate surfaces. Firstly, effect of three different medium including basal medium Dulbecco's modified Eagle's medium (D-MEM) containing L-glutamine, L-glutamine + 2mM a-ketoglutarate and L-alanyl-glutamine (GlutaIbMAXTM) on M2139 clone was investigated. The kinetics of cell grew in basal medium containing L-glutamine + a-ketoglutarate condition was similar to cells grown on DMEM-GlutaMAX containing medium whereas D-MEM containing Lglutamine showed lowest productivity. With the maximal viable cell density (6.85 x 10⁶ cells/ml) and highest specific mAb production rate (3.9 ug/ml.10⁴ viable cell. day), DMEM- GlutaMAX was suitable for culture M2139 cell line. Secondly, the effect of aketoglutarate on growth and mAb production was studied in an anchorage independent and anchorage dependent hybridoma cell lines, D2 and 2C83G2 clone. Both hybridomas were seeded into serum free and serum containing media and cultured for 8 days. caketoglutarate was added in both media at initial and after 72 h of cultivation The results indicate that a-ketoglutarate can enhance hybridoma cell growth but depend on hybridoma clone and the media used. The last section of this study was focused on process development including the sterilization technique and design of cryogel reactors. Heat sterilization was compared to sterilization by alcohol. Heat sterilization by autoclave at 121 °C for 15 min of the gelatincryogel monolith has an affect on color and decreased cell binding capacity. But it controlled contamination better than 70% alcohol treatment. In order to increase productivity, three designs of cryogel reactor were compared. This included a 350 cm³ (0 4.5 x 21 cm) cryogel monolith reactor, cryogel disc reactor and macroporous gel particles (MGPs) in packed bed reactors. The 350 cm³ (0 4.5 x 21 cm) cryogel monolith reactor have a major problem of nutrient shortage and oxygen transfer. The cryogel discs were produced by first preparing cryogel in sheet form in A4 size aluminum trays. The cryogel sheet was then cut in disc type (4) 4.5 x I cm) and stacked into a glass column reactor (0 4.5 x 21 cm). The cryogel disc reactor had a problem with gel discs floating up while the solution was circulated. The quality of the cryogel sheet was also difficult to control. The most effective design was the macroporous gel particle (MGP) in a packed-bed reactor. The MGPs were made of supermacroporous gels and modified with gelatin which was housed in plastic carriers (PC; 7 x 9 mm). Cell proliferation and long term production of monoclonal antibody IgG2b by M2139 hybridoma cells immobilized in macroporous gel particles (MGPs) in packed-bed reactor were studied for a period of 60 days. Cells were trapped in the interior part of MGPs by attaching to the void space of the gel matrix as three-dimensional (3-D) cultivation using gelatin as a substrate layer. Cells in MGPs were able to grow and secrete antibody for 30 days. A medium reservoir then replaced with a fresh one. The mAb concentrations reached 180 ug/ml with productivity of 6ug/ml per day from 30 days continuous nun in the packed-bed reactor. After being supplied with fresh medium, cells again showed continuous growth for another 30 days with mAb production efficiency of 50%. These results demonstrate that MGPs can be used efficiently as supporting carrier for long-term monoclonal antibody production. In addition, MGPs packed-bed reactor can solve problem of oxygen and nutrient transfer, decrease in the lag phase time and increase in productivity. In summary, a supermacroporous cryogels has been found to be very useful as a supporting material for productive hybridoma cell culture. Cells were found to be viable inside porous matrix of the cryogel during the study period and secreted antibodies continuously. The antibodies thus obtained from the cryogel reactor were found to be functionally active in vivo, as demonstrated by their capacity to induce arthritis in mice. This research demonstrated that a supermacroporous cryogel promising potential as a novel bioreactor allowing 3-D cultivation and long term mAb production. |
| Year | 2007 |
| Corresponding Series Added Entry | Asian Institute of Technology. Dissertation ; no. FB-07-02 |
| Type | Dissertation |
| School | School of Environment, Resources, and Development (SERD) |
| Department | Department of Food, Agriculture and Natural Resources (Former title: Department of Food Agriculture, and BioResources (DFAB)) |
| Academic Program/FoS | Food Engineering and Bioprocess Technology (FB) |
| Chairperson(s) | Rakshit, Sudip Kumar |
| Examination Committee(s) | Athapol Noomhorm;Yakupitiyage, Amararatne;Kumar, Ashok;Roychoudhury, Pradip Kumar |
| Scholarship Donor(s) | Valaya-Alongkorn Rajabhat University;RTG Fellowship |
| Degree | Thesis (Ph.D.) - Asian Institute of Technology, 2007 |